Conditions for Long-Term Culture of Cattle Undifferentiated Spermatogonia
Conditions for Long-Term Culture of Cattle Undifferentiated Spermatogonia
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DOI：
10.1095/biolreprod.116.139832
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发表时间：
2016-07-01
影响因子：
3.6
通讯作者：
Oatley, Jon M.
中科院分区：
文献类型：
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作者：
Oatley, Melissa J.;Kaucher, Amy V.;Oatley, Jon M.
持续而强大的精子发生依赖于含有干细胞的未分化精原细胞群的作用。精原干细胞（SSC）的一个显着特征是从供体睾丸分离并移植到允许的受体睾丸后能够再生精子发生。这种能力作为增强牲畜繁殖能力的工具具有巨大潜力，可以提高生产效率。由于 SSC 是未分化精原细胞群体中的一个罕见子集，因此从供体睾丸组织中分离出来后，一段时间的体外数量扩增至关重要。在这里，我们描述了从青春期前公牛睾丸中分离细胞组分的方法，该细胞组分富含未分化的精原细胞，并在饲养细胞共培养和无饲养细胞格式中长期维持细胞。为了实现这种方法，我们衍生了牛胎儿成纤维细胞（BFF）作为饲养者，以优化培养基条件，促进牛未分化精原细胞维持至少2个月。此外，我们设计了一种采用 BFF 条件培养基的无饲养层系统，可在体外维持牛未分化精原细胞至少 1 个月。可以对所描述的方法进行优化，以提供牛 SSC 指数扩增的平台，从而提供移植到受体睾丸中所需的数量。
Continual and robust spermatogenesis relies on the actions of an undifferentiated spermatogonial population that contains stem cells. A remarkable feature of spermatogonial stem cells (SSCs) is the capacity to regenerate spermatogenesis following isolation from a donor testis and transplantation into a permissive recipient testis. This capacity has enormous potential as a tool for enhancing the reproductive capacity of livestock, which can improve production efficiency. Because SSCs are a rare subset of the undifferentiated spermatogonial population, a period of in vitro amplification in number following isolation from donor testicular tissue is essential. Here, we describe methodology for isolation of a cell fraction from prepubertal bull testes that is enriched for undifferentiated spermatogonia and long-term maintenance of the cells in both the feeder cell coculture and the feeder-free format. To achieve this method, we derived bovine fetal fibroblasts (BFF) to serve as feeders for optimizing medium conditions that promote maintenance of bovine undifferentiated spermatogonia for at least 2 mo. In addition, we devised a feeder-free system with BFF-conditioned medium that sustained bovine undifferentiated spermatogonia for at least 1 mo in vitro. The methodologies described could be optimized to provide platforms for exponential expansion of bovine SSCs that will provide the numbers needed for transplantation into recipient testes.